Development of a SARS-CoV-2-derived receptor-binding domain-based ACE2 biosensor

Abstract

The global outbreak of coronavirus disease and rapid spread of the causative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent a significant threat to human health. A key mechanism of human SARS-CoV-2 infection is initiated by the combination of human angiotensin-converting enzyme 2 (hACE2) and the receptor-binding domain (RBD) of the SARS-CoV-2-derived spike glycoprotein. Despite the importance of these protein interactions, there are still insufficient detection methods to observe their activity at the cellular level. Herein, we developed a novel fluorescence resonance energy transfer (FRET)-based hACE2 biosensor to monitor the interaction between hACE2 and SARS-CoV-2 RBD. This biosensor facilitated the visualization of hACE2-RBD activity with high spatiotemporal resolutions at the single-cell level. Further studies revealed that the FRET-based hACE2 biosensors were sensitive to both exogenous and endogenous hACE2 expression, suggesting that they might be safely applied to the early stage of SARS-CoV-2 infection without direct virus use. Therefore, our novel biosensor could potentially help develop drugs that target SARS-CoV-2 by inhibiting hACE2-RBD interaction.

Keywords: ACE2; Biosensor; CQ, chloroquine; FRET; HCQ, hydroxychloroquine; Live-cell imaging; NA, numerical aperture; RBD, receptor-binding domain; RBM, receptor-binding motif; ROI, region of interest; SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SEM, standard error of the mean; bg, background; hACE2, human angiotensin-converting enzyme 2.

Fig. 1

Design and characterization of the FRET-based ACE2 biosensor. (a) Schematic diagram of the ACE2 biosensor. The ACE2 biosensor is composed of a YPet acceptor, SARS-CoV-2-derived RBD domain, and truncated Turquoise2-GL donor. The ACE2 biosensor mutants are E484 P (positive control) and F486 L (negative control). The ACE2-WT truncated membrane localization signal was inserted into the pmScarlet-I vector, and the ACE2-M82 K construct was developed as a negative control. (b) The ACE2 biosensor shows the conformational change by binding to ACE2, leading to an increase in the FRET ratio. This diagram was produced by Biorender (http://biorender.com/) (c) The normalized FRET/CFP emission ratios of the ACE2 biosensors (WT, E484 P, and F486 L) in HEK293 T cells co-transfected with ACE2-WT, ACE2-M82 K, or control pmScarlet-I vector (****p < 0.0001).

Fig. 2

Dynamic visualization of the ACE2 biosensor in response to the ACE2 inhibitor. (a) Time-lapse FRET/CFP ratio images of the ACE2 biosensors (WT, E484 P, and F486 L) in HEK293 T cells co-transfected with control, ACE2-WT, or ACE2-M82 K treated with 100 μM HCQ. Hot and cold colors indicate high and low affinities between ACE2s and RBDs, respectively (scale bar = 10 μm). (b) Time courses of the normalized FRET/CFP ratio of the ACE2 biosensor-WT in HEK293 T cells co-transfected with ACE2-WT (red, n = 4), control (blue-violet, n = 5), or ACE2-M82 K (turquoise, n = 9) treated with 100 μM HCQ. All error bars represent SEM. (***p < 0.001). (c) Time courses of the normalized FRET/CFP ratios of the ACE2 mutant biosensors (E484P-positive control and F486L-negative control) in HEK293 T cells co-transfected with ACE2-WT or ACE2-M82 K treated with 100 μM HCQ (red, blue-violet, and turquoise; n = 5,5, and 4, respectively). All error bars represent SEM (***p < 0.001).

Fig. 3

Validation of the ACE2 biosensor in response to MLN-4760, a specific ACE2 inhibitor. (a) Time-lapse FRET/CFP ratio images of the ACE2 biosensor in HEK293 T co-transfected with ACE2-WT treated with DMSO, 0.5 nM, and 30 nM MLN-4760, respectively. Hot and cold colors indicate high and low affinities between exogenous ACE2 and RBD, respectively (scale bar = 10 μm). (b) Time courses of the normalized FRET/CFP ratios of the ACE2 biosensor in HEK293 T co-transfected with ACE2-WT treated with DMSO, 0.5 nM, and 30 nM MLN-4760 (gray, blue, and red; n = 4,4, and 7, respectively). All error bars represent SEM (****p < 0.0001).

Fig. 4

ACE2 biosensor specificity in SY5Y and HepG2 cells. (a-b) Time-lapse CFP, FRET, DIC, and FRET/CFP ratio images of the ACE2 biosensor in SH-SY5Y and HepG2 cells treated with 100 μM HCQ, respectively. Hot and cold colors indicate high and low endogenous ACE2 and RBD affinities, respectively (scale bar = 10 μm). (c) Time courses of the normalized FRET/CFP ratios of the ACE2 biosensor in SH-SY5Y (blue-violet, n = 7) and HepG2 (red, n = 10) cells treated with 100 μM HCQ. All error bars represent SEM (****p < 0.0001).